| Objective:1.Through network pharmacology to study the main pharmacodynamic substances of Astragalus-Angelica in combination with anti-vascular intimal hyperplasia and their possible mechanism;2.Based on the results of network pharmacological research,to study the effects of different compatibility ratios of Astragalus-Angelica on vascular intimal hyperplasia,and to explore the effects of Astragalus-Angelica compatibility on the phenotypic transformation and proliferation of Vascular smooth muscle cells(VSMC)in vascular intimal hyperplasia models,Vascular Adventitia Fibroblasts cells(VAF)activation,and study its mechanism of action from PI3K/Akt and TGF-β/Smad signaling pathways.Methods:1.The TCMSP database and the Pharmmaper database were used to obtain the effective chemical components and targets of Astragalus and Angelica respectively;the multi-disease database(including Genecards,Dig Se E and OMIM databases)were used to retrieve and collect disease targets related to vascular intimal hyperplasia;the targets between the disease and the drug were screened out as predictive targets of the action of the drug component;the string software was used to obtain the relationship between the predicted target proteins,and the core targets were screened out according to the size of the relationship.Cytoscape3.6.1 software was used to establish the "drug-active ingredient-disease-target" network and the core targets interaction network;GO biological process analysis and KEGG pathway enrichment analysis were performed on the selected core targets.2.Clean-grade New Zealand white rabbits,half of gender,were randomly divided into sham operation group,model group,Astragalus group,Angelica group,Astragalus-Angelica 1:1 group,Astragalus-Angelica 1:5group,and Astragalus-Angelica 5:1 group,Atorvastatin calcium group.The rabbit vascular intimal hyperplasia model was established by common carotid artery cannula combined with high-cholesterol diet.The sham operation group only performed separation operation without cannulation.On the first day after the operation,the sham operation group was fed with ordinary diet,and the other groups were fed with high cholesterol diet(2%cholesterol + 3% olive oil + 95% ordinary feed);At the same time,each medication group was given corresponding drugs by intragastric instillation,and the sham operation group The model group was given distilled water intragastric instillation for 28 days.After the experiment,blood was collected from the abdominal aorta,the plasma total triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)level were detected by enzymatic method;Masson staining and the morphometric method was used to measure the intimal area(IA),intimal Membrane thickness(IT),media area(MA),media thickness(MT),adventitia area(AA),adventitia thickness(AT),hyperplasia ratio of intimal thickness(HRIT),and hyperplasia ratio of intimal area(HRIA);immunohistochemical was used to detect the common carotid artery intima proliferating cell nuclear antigen(PCNA),osteopontin(OPN),α-smooth muscle actin(α-SMA)protein expression,to reflect the phenotypic transformation,migration,and proliferation of smooth muscle cells;Western blot was used to determinate the phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)related pathway protein expression to reflect the mechanism of VSMC phenotypic transformation;Immunohistochemical method was used to detect the expression of local pro-inflammatory factors including Tumor necrosis factor α(TNF-α)and interleukin-1 beta(IL-1β)in damaged blood vessels to reflect the influence on the intimal hyperplasia of blood vessels;Immunofluorescence double labeling method was used to mark the myofibroblast marker α-SMA and the fibroblast marker vimentin to reflect the proliferation and activation of outer membrane fibroblasts;Western blot method was used to detect the expression of transforming growth factor-1(TGF-β1),Smad2 and p-Smad2 in the signaling pathway related to VAF activation in the injured common carotid artery.Results: 1.A number of 20 active ingredients was collected of the Astragalus-Angelica,and a total of 193 potential drug targets and 487 potential disease targets were obtained,which mainly act on multiple targets such as EGFR,ESR1,ALB,MAPK8,PGR,etc.PI3K/Akt,MAPK,TGF-β/Smad,Ras and many other signal pathways.2.(1)In the rabbit vascular intimal hyperplasia model,the compatibility of Astragalus and Angelica has a certain lipid-regulating effect.Astragalus-Angelica1:1 and Astragalus-Angelica5:1 groups can significantly increase plasma HDL-C level,and reduce plasma TC,TG,LDL-C level.(2)In the rabbit vascular intimal hyperplasia model,pathological examination showed that obvious irregular intimal hyperplasia was seen in the injured common carotid artery in the model group,the lumen was significantly narrowed,and a large number of vascular smooth muscle cells(VSMC)existed in the hyperplastic intima,disorderly arrangement.The morphometric analysis showed that compared with the model group,the IA,IT,HRIA and HRIT of the Astragalus-Angelica1:1 and5:1 groups were significantly reduced,and the degree of vascular intimal hyperplasia was significantly reduced.(3)Immunohistochemical detection showed that the expression of α-SMA in the hyperplastic intima of the model group was decreased,and the expression of OPN and PCNA was up-regulated.Compared with the model group,the expression of α-SMA was up-regulated in the Astragalus-Angelica1:1 and 5:1 compatibility group,while the expression of OPN and PCNA was down-regulated;As the Astragalus and Angelica alone had no significant effect on the expression ofα-SMA,OPN and PCNA(P > 0.05).It indicates that the compatibility of Astragalus and Angelica can inhibit the phenotype transformation and proliferation of smooth muscle cells.(4)PI3K/Akt signaling pathway related protein detection showed that the expression of PI3 K and Akt protein in each group did not change significantly,while the expression of activated form of p-PI3 K and p-Akt increased significantly in the model group;compared with the model group,the expression of p-PI3 K and p-Akt protein in the Astragalus-Angelica1:1 and 5:1 groups were significantly reduced.It indicates that the compatibility of Astragalus and Angelica can inhibit the activation of PI3K/Akt signaling pathway in the vascular intimal hyperplasia model.3.(1)In the rabbit vascular intimal hyperplasia model,the adventitia of the model group increased significantly(P<0.05).The adventitia of the injured common carotid artery in Astragalus-Angelica1:1 group and 5:1 group also had different degrees of hyperplasia,but compared with the model group,the degree of adventitia hyperplasia was significantly reduced(P<0.05).It is suggested that the compatibility of Astragalus and Angelica can inhibit the proliferation of adventitia of blood vessels.(2)The expression of α-SMA and Vimentin in damaged blood vessels in the model group increased significantly(P<0.05),suggesting that in the vascular proliferation model,VAF transforms into myofibroblasts and promotes intimal hyperplasia.The expression of α-SMA and Vimentin decreased in Astragalus-Angelica1:1group,5:1 group and Atorvastatin calcium group.It shows that the compatibility of Astragalus and Angelica can inhibit the transformation of rabbit vascular intimal hyperplasia model from VAF into myofibroblasts.(3)Local pro-inflammatory factors TNF-α and IL-1β in the vascular intimal of the model group were significantly increased;compared with the model group,the Atorvastatin calcium group and the Astragalus-Angelica1:1,5:1groups the expression of TNF-α and IL-1β were significantly reduced;there was no significant difference in the expression of TNFα and IL-1β in the inner membrane of the injured area between the Astragalus-Angelica1:1,5:1and the Atorvastatin calcium groups.It shows that the compatibility of Astragalus and Angelica can inhibit the expression of local inflammatory factors in the blood vessel of the vascular intimal hyperplasia model and reduce the local inflammation.(4)The expression of TGF-β1,Smad2,p-Smad2 in the model group was significantly higher than that in the sham operation group;Astragalus-Angelica1:1 group,Astragalus-Angelica5:1group and Atorvastatin calcium group could significantly reduce TGF-β,Smad2,and p-Smad2 expression.It is suggested that the compatibility of Astragalus and Angelica can inhibit the activation of TGFβ/Smad2 signaling pathway in the vascular intimal hyperplasia model.Conclusions: 1.Through network pharmacology research,it is predicted that the anti-vascular intimal hyperplasia effect of Astragalus-Angelica may have 20 active ingredients,which may act on 193 potential drug targets,and the main effects involve Multiple signaling pathways such as PI3K-Akt,MAPK,TGFβ/Smad and Ras.It provides a basis for further research on the pharmacodynamic substances and mechanism of improve vascular intimal hyperplasia of Astragalus-Angelica combination.2.The compatibility of Astragalus-Angelica can inhibit vascular intimal hyperplasia,and the compatibility of Astragalus-Angelica1:1 and 5:1groups have obvious effects.3.The compatibility of Astragalus-Angelica1:1 group and 5:1 group can reduce plasma TC,TG,LDL-C levels,and increase HDL-C levels,indicating that its anti-vascular intimal hyperplasia effect is related to its lipid-regulating effect.4.Astragalus-Angelica1:1 group and 5:1 group can inhibit the phenotype transformation and proliferation of VSMC in the intimal hyperplasia model,and its mechanism of action is related to the inhibition of PI3K/Akt signaling pathway activation.5.Astragalus-Angelica1:1 group and 5:1 group can play an anti-vascular intimal hyperplasia effect by inhibiting VAF activation and reducing local inflammation.Its mechanism of action is related to inhibiting the activation of local vascular TGF-β/Smad2 signaling pathway. |
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